This project is in the area of biochemistry and addresses research into prevention of protein aggregation-based diseases such as Alzheimer’s. In cells, molecular chaperones assist with correcting protein folding to prevent diseases caused by misfolded protein aggregation. Cochaperones stimulate and enhance chaperone activity by facilitating client protein presentation and ATP hydrolysis. Heat shock proteins (Hsps) are a major class of chaperones that are present in most organisms and have homologues across different organisms, cells, and organelles. Grp78, a Hsp homologue in the human endoplasmic reticulum (ER), is not well represented in chaperone research since it is difficult to purify from induction in bacteria cells. The human ER cochaperone ERdj6 is slightly easier to purify than Grp78, and this allows us to combine it with the well-characterized bacterial chaperoning system to develop a model for Grp78 chaperoning mechanisms and client proteins. This study used E. coli chaperone DnaK, E. coli cochaperone DnaJ, and ERdj6 to create this model. Each cochaperone was paired with DnaK and tested for activity with ATPase assays. The assays show that the experimental DnaK-ERdj6 pair had similar levels of activity stimulation to the wild type DnaK-DnaJ pair, with the DnaJ pair showing peak activity at a lower concentration relative to DnaK than that of the ERdj6 pair. Both pairs show significantly greater activity than the DnaK, DnaJ, and ERdj6 controls. This shows that the DnaK-ERdj6 pair is likely a suitable model for characterizing ER chaperoning mechanisms. Model confirmation using a protein aggregation assay study will allow us to use the model to characterize protein clients of Grp78. This research has been important towards my future intentions in protein research as it has provided me with experimental methods and with additional background knowledge for future research.
Authors: Tim Miller, Erin Unruh, Andrea Kravats PhD.
Faculty Advisor: Andrea Kravats, Chemistry and Biochemistry
Graduate Student Advisor: Erin Unruh, Chemistry and Biochemistry
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